Genetic sequence information and measurements of gene expression, DNA copy number, DNA methylation, and the like, have applications in functional genomic research, disease diagnostics, drug discovery, and the like. The collection of genetic information and information pertaining to gene expression has been facilitated by the development of microarray technologies. These technologies often involve imaging a microfabricated array of, e.g., nucleic acid (probe) sequences disposed on a support, such as a microchip or slide.
Because of the small feature (e.g. spot) size and more compact architecture, high density arrays typically require high resolution optical devices or readers to interrogate the sites of the arrays. Several vendors have manufactured microchip reading devices to detect the signals from these chips. These devices are generally based on fluorescence detection; i.e., the detection of fluoresced light resulting from one or more fluorophores upon exposure of the fluorophores to an excitation light. They typically employ either confocal scanners combined with photomultiplier tube (PMT) detectors (for companies such as Genetic MicroSystems, GSI Lumonics, Virtek Vision), or a CCD detector with an imaging lens (Hitachi). These devices can typically detect two or more dyes by alternating the excitation laser wavelength to match a specific dye excitation spectral maximum, and/or switching an optical filter to match the fluorescent spectrum of a particular dye.
Microarray measurements can be very precise, being limited fundamentally by the counting statistics of the binding of the labeled molecules to the array spots, or by the collection of sufficient photons from the fluorochromes bound to the spots. In practice the signal intensities are usually higher than these fundamental limits, and the predominant noise is supplied by characteristics of the microarray experiment, including non-specific binding to array spots, background binding to the array substrate, etc.
Array measurements also result in signals that can range in intensity over many orders of magnitude in a single experiment. Thus the number of mRNA molecules of different sequences in a cell population may range in abundance by a factor of 10,000 or more. If one wants to accurately compare the relative abundances of these different species, as is the goal of microarray-based gene expression or DNA copy number measurements, the hybridization intensities need to be measured with high accuracy over this wide dynamic range.
Two types of optical detectors, photomultiplier tube (PMT) and charged coupled devices (CCD), are commonly used in microarray imaging systems at the current time. In PMT-based systems, a point source of light, for example a focused laser beam, is scanned over the array, causing emission of light from the array. The emitted light is detected by the PMT and converted to an electrical current, and an image of the array is built up by associating the output of the PMT with the position of the scanning beam as it moves over the array. In common CCD systems the entire array, or portion thereof, is illuminated and the emitted light is imaged onto the CCD chip. Thus light is quantitatively measured from multiple points of an array simultaneously. CCD systems have potential advantages over PMT systems in several major areas: 1) The output of a CCD is linearly proportional to light intensity over a wider dynamic range than a PMT. 2) The efficiency of detecting light (quantum efficiency) is higher. 3) The mechanical design is simpler since it is not necessary to scan the illumination beam.
In order to obtain the benefits of CCD imaging, one needs to overcome several significant problems in optical design. These include minimizing or properly correcting for residual spatial variations in the sensitivity of the imaging system over the surface of the array, design of filters to obtain adequate spectral discrimination of multiple wavelengths and to reduce stray light, and reduction of “ghost” images due to reflections within the optical system.